The Enterococcus faecalis plasmid pAD1 is the prototype of a group of plasmids native to this organism which possess a unique mechanism of conjugative transfer. These plasmids respond to specific peptide sex pheromones produced by potential recipients by directing the synthesis of an adhesive substance which facilitates the formation of mating aggregates. While much has been learned about the mechanisms of pheromone-inducible conjugation, little is known about the maintenance functions of large, low copy number plasmids native to gram-positive bacteria in general. The goal of this project is to identify and characterize the plasmid-encoded determinants necessary for replication, copy number control, and stable inheritance of pAD1. The pAD1 maintenance region has been isolated on a 5 kilobase (kb) piece of plasmid DNA and transposon mutagenesis has identified several determinants involved in plasmid maintenance. In addition, an incompatibility determinant has been cloned which maps to a portion of the maintenance region believed to be involved in copy number control. As the first step in characterizing the pAD1 maintenance region, the DNA sequence will be identified. Potential protein binding sites will be isolated and tested for their ability to function as incompatibility determinants and to bind replicon- encoded proteins in gel retardation assays. Possible roles of individual determinants, suggested by sequence homology with known maintenance functions of other plasmids of by mutagenesis experiments, will be examined by a variety of approaches including i) separation of origin and initiator functions by cloning, ii) study of incompatibility characteristics of copy control functions, and iii) stabilization of heterologous replicons by suspected stability determinants.